We maintain that a thorough and rigorous analysis of trafficking of EGFR ligands in polarized epithelial cells will continue to provide important insights into basic cell biology and the pathogenesis of colorectal cancer (CRC). The current grant cycle has been a period of significant productivity with 39 manuscripts and 4 reviews published that cite this grant. Simply stated, we intend to demonstrate that in the colon TGFA is beneficial and EREG is harmful. Endogenous TGFA acts to maintain colonic homeostasis; it stabilizes NKD2 to ensure its efficient delivery to the basolateral surface. After delivering TGFA to the basolateral surface, NKD2 binds and degrades DVL-1 (a positive regulator of Wnt signaling) so as to maintain tight regulation of canonical Wnt signaling. Loss of NKD2 is a frequent late event in CRC, and induction of NKD2 in Caco-2 cells initiates a program of goblet cell differentiation. Whereas TGFA mRNA is decreased in CRC, EREG expression is increased. Overexpression of EREG in colon cancer has been linked to metastasis. We show that tyrosine (Y)156 in the cytoplasmic tail of EREG regulates its basolateral trafficking and have identified multiple interacting partners. Remarkably, EREG(Y156A) is completely missorted to the apical surface of polarized MDCK cells, and the cells are morphologically and functionally transformed. We propose two aims to elucidate the divergent roles of TGFA and EREG in normal colon and CRC. Aim 1. Test the hypothesis that NKD2 acts as a tumor suppressor. We will elucidate the mechanism by which induction of NKD2 in Caco-2 cells results in goblet cell differentiation. Using mice we recently generated, we will determine whether loss of NKD2 in Wnt-dependent (AOM) and Wnt-independent (Smad3-/-) colonic tumors results in more advanced tumors. We also propose to examine post-transcriptional regulation of NKD2 as part of a long-term therapeutic strategy to treat CRC by regulated re-expression of NKD2. Aim 2. Identify factors (and their underlying mechanisms of action) that regulate EREG basolateral trafficking and signaling. Using wild-type EREG and EREG (Y156A), we propose to elucidate mechanisms underlying the invasive phenotype of apically redirected EREG. Finally, we will test the hypothesis that EREG is a metastasis promoter by attempting to prevent lung metastasis in a syngeneic mouse colon cancer model through elimination of EREG.